The invention relates to cell populations and methods of producing them.
There is strong empirical evidence that during murine embryogenesis a common precursor to hematopoietic and endothelial cells exists. A pluripotent precursor cell, caled the hemangioblast, which carries this bipotential, was first hypothesized in 1900 by W. His. Putative hemangioblasts have been teased out of embryonic cultures and manipulated by cytokines to differentiate along either hematopoietic or endothelial pathways. Using population kinetics it has been demonstrated that pre-endothelial/pre-hematopoietic cells in the embryo clearly arise out of a phenotype CD 34xe2x88x92 population. (Choi et al., A Common Precursor for Hematopoietic and Endothelial Cells, Development 125, 725-732 (1998).)
Additionally, it has recently been found that sheep can be successfully engrafied in utero with adult human CD34xe2x88x92, Linxe2x88x92 cells, resulting in long-term engraftment and multi-lineage hematopoietic cell/progenitor expression. Significant numbers of human CD34+ cells were detected in the animals that were transplanted with the CD34xe2x88x92, Linxe2x88x92 cells. The resulting conclusion was that the CD34xe2x88x92 fraction of normal human bone marrow contains cells capable of enabling in utero engraftment, possibly through the differentiative production of engraftable CD34+ cells in the fetal microenvironment. (Zanjani et al., Human Bone Marrow CD34xe2x88x92 Cells Engraft In Vivo and Undergo Multilineage Expression That Includes Giving Rise to CD34+ Cells, Experimental Hematology 26:1-221 (1998).)
The invention provides enriched stem cell containing populations that can be expanded, and thus can be of great value to patients in need of cellular therapy, e.g., cancer therapy, immunotherapy, and gene therapy.
Preferred populations include at least 5% human non-fetal uncommitted hemangioblasts, i.e., common precursors of hematopoietic cells and endothelial cells; by xe2x80x9cuncommittedxe2x80x9d it is meant that the hemangioblasts are not yet committed to either lineage, i.e., under the proper conditions the cells can become either hematopoietic cells or endothelial cells. These hemangioblasts are stable, not transient, and are present in the tissue of fully developed individuals, such as in newborn infants and adults. We have, in fact, discovered that these hemangioblasts can be isolated from cord blood following birth. The presence of hemangioblasts in non-embroid tissue was unexpected and presents novel opportunities.
As shown schematically in FIG. 1, the uncommitted hemangioblasts can be stimulated to become hematopoietic cells or endothelial cells, by selecting appropriate growth factors in which to expand the population, as will be discussed in detail below. Thus, when supplied with one cocktail of growth factors, these hemangioblasts can be amplified (i.e., the number of hemangioblasts can be increased), and/or they can be differentiated to provide a supply of hematopoietic cells, for example to patients who are immune compromised or require gene therapy with hematopoietic cells. When supplied with a different cocktail, the hemangioblasts can be amplified and/or can be differentiated to become endothelial cells, useful for example in wound healing, e.g., healing of slow or non-healing diabetic sores. The endothelial cells can also be transfected ex vivo, e.g., with genes which produce angiogenic factors, and used in gene therapy, for example to stimulate angiogenesis in patients with vascular or cardiac insufficiency. Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. Ojeifo, et al., Cytokines Mol Ther 1996 Jun;2(2):89-101.
Populations which have been expanded to contain a significant percentage of these uncommitted hemangioblasts will provide a high level of engraftment while starting from a relatively small sample, since the uncommitted hemangioblasts can be stimulated to become hematopoietic cells by supplying them with appropriate growth factors.
Accordingly, in one aspect, the invention features a method for providing a cell population containing non-fetal hemangioblasts. The method includes (a) providing a first cell population containing non-fetal hemangioblasts; and (b) growing the first cell population under conditions that promote the proliferation of the non-fetal hemangioblasts. The invention also features cell populations formed by expansion of a population containing non-fetal hemangioblasts.
Preferred embodiments of the invention include one or more of the following features. In the growing step (step (b), above), the conditions are such that the number of said non-fetal hemangioblasts and their proximity to each other are sufficient to increase the proportion of non-fetal hemangioblasts in the population. The method includes, prior to the growing step, enriching the first cell population for non-fetal hemangioblasts. The method also includes separating the non-fetal hemangioblasts from other cells in the cell culture, e.g., by a negative selection process. The separating step is performed concurrently with, intermittently during, or following, the growing step. The separating step is performed more than once during cell proliferation, e.g., every 5 to 10 days. The growing step includes providing at least one growth factor, more preferably a cocktail of growth factors, to the cell population during cell proliferation. At least some of the non-fetal hemangioblasts, preferably at least 2%, more preferably at least 5%, more preferably at least 15% and most preferably at least 25%, are uncommitted human hemangioblasts. At least some of the non-fetal hemangioblasts, preferably at least 2%, more preferably at least 5%, more preferably at least 15% and most preferably at least 25%, are CD 34xe2x88x92, Linxe2x88x92 cells. The percentage of cells that are CD 34xe2x88x92, Linxe2x88x92 and/or are uncommitted human hemangioblasts is higher in the enriched cell culture than in the starting cell culture. The uncommitted human hemangioblasts are characterized as: CD2xe2x88x92, CD3xe2x88x92, CD 14xe2x88x92, CD16xe2x88x92, CD19xe2x88x92, CD24xe2x88x92, CD56xe2x88x92, CD66bxe2x88x92, glycophorin Axe2x88x92. The uncommitted human hemangioblasts are further characterized as: flk-1+, CD45+, CXCR4+, MDR+ (Pgp).
In another aspect, the invention features an enriched cell population comprising non-fetal hemangioblasts, the enriched cell population resulting from expansion of a starting cell population containing fewer non-fetal hemangioblasts than the enriched cell population.
Preferred embodiments include one or more of the following features. The starting cell population contains at least 10% fewer non-fetal hemangioblasts than the enriched cell population. The percentage of cells that are non-fetal hemangioblasts in the enriched cell culture is the same as or higher than the percentage of cells that are non-fetal hemangioblasts in the starting cell culture.
The invention also features a composition of cells in which at least 2%, more preferably at least 5%, more preferably at least 15%, and most preferably at least 25% of the cells are non-fetal hemangioblasts, and methods of making such a composition. Preferably the non-fetal hemangioblasts are human uncommitted hemangioblasts that are Linxe2x88x92 cells and are characterized as: CD2xe2x88x92, CD3xe2x88x92, CD14xe2x88x92, CD16xe2x88x92, CD19xe2x88x92, CD24xe2x88x92, CD56xe2x88x92, CD66 bxe2x88x92, glycophorin Axe2x88x92, flk-1+, CD45+, CXCR4+, MDR+.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.